摘要
研究背景:骨肉瘤是最常见的原发于骨组织的恶性肿瘤,好发于儿童及青少年,其恶性程度高,易复发和转移,预后较差。治疗方法包括手术治疗、化疗、放射治疗及综合疗法,新辅助化疗的应用使骨肉瘤的治愈率有了很大的提高,但临床治疗,特别是对于多药耐药(multidrug resistance MDR)病例的治疗仍存在许多难题。探索新的、有效的化疗药物将有助于骨肉瘤的治疗。
姜黄(Curcuma)是一种常用中药,它广泛应用于食品上色和佐味剂。从姜黄中提取的酚性色素——姜黄素(Curcumin,Cur)是姜黄的主要有效成份,姜黄具有多方面的药理作用,如抗炎、抗氧化、抗凝、降血脂、抗动脉粥样硬化及抗肿瘤等作用,尤其是作为一种具有良好发展前景的抗癌药物,其抗肿瘤作用已日益引起人们的重视,成为近年来研究的热点。姜黄素的抗癌机理是多方面的,其中诱导肿瘤细胞凋亡的机制受到人们的广泛关注。有研究表明,姜黄素能抑制多种体外培养的肿瘤细胞如前列腺癌、鼻咽癌、胃癌、结肠癌、肝癌等的生长及增殖,并诱导其凋亡,且可逆转或部分逆转这些肿瘤细胞的P-糖蛋白(P-gp)介导的多药耐药。化疗在恶性肿瘤的治疗中具有非常重要的作用,而耐药性是化疗失败的最常见和最难以克服的问题之一。70年代中期,Juliano等人发现了肿瘤的多药耐药(MDR)现象,即对一种药物耐药的肿瘤,同时对另外一些与之化学结构和作用机制完全不同的药物也有交叉耐药现象。因此,揭示肿瘤产生多药耐药性的机理及其与临床化疗之间的关系成为人们研究的热点。但姜黄素对人骨肉瘤细胞上述方面作用的研究尚未见报道。
本研究采用MTT比色法检测入骨肉瘤细胞U-20S的增殖;Hoechst33258染色后荧光显微镜法观察U-20S细胞凋亡的形态学改变,Annexin V-FITC/PI双染色,流式细胞术检测细胞凋亡;Caspase活性定量检测试剂盒检测半胱天冬特异性蛋白酶-9,8,3(Caspase-9,8,3)的活性,探讨姜黄素对体外培养的人骨肉瘤细胞增殖、凋亡的影响与可能机制,并进一步探讨姜黄素对人骨肉瘤U-20S耐药细胞U-20S/ADM中多药耐药的逆转作用。为临床上骨肉瘤的治疗提供新的理论与方法。
第一部分姜黄素对人骨肉瘤U-20S细胞的作用研究
目的:观察姜黄素对人骨肉瘤U-20S细胞增殖、凋亡的影响,探讨姜黄素对人骨肉瘤的作用。
方法:四甲基偶氮唑盐(MTT)比色法检测U-20S细胞的增殖;Hoechst 33258染色后荧光显微镜法观察U-20S细胞凋亡的形态学改变,并计算凋亡核百分率;Annexin V-FITC/PI双染色,流式细胞术检测细胞凋亡;Caspase活性定量检测试剂盒检测半胱天冬特异性蛋白酶-9,8,3(Caspase-9,8,3)的活性。
结果:
1.姜黄素可显著抑制U-20S细胞的体外增殖,并呈浓度和时间依赖性。
2.Hoechst 33258染色显示U-20S细胞经姜黄素(20μmol/L)分别处理24h和48h后,细胞出现凋亡,形态学变化表现为凋亡细胞体积缩小,核固缩,镜下细胞核呈致密浓染或碎块状致密浓染。FACS检测结果显示,20μmol/L姜黄素处理12h、24h、48h后的凋亡细胞数分别为13.81±1.07%、27.03±2.32%、40.76±3.96%。U-20S细胞分别经10μmol/L、20μmol/L、40μmol/L姜黄素处理48h后,凋亡细胞数逐渐增多,凋亡核百分率计数分别为(11.26±3.03)%,(32.15±2.88)%,(55.24±4.40)%。FACS检测结果显示,10μmol/L、20μmol/L、40μmol/L姜黄素处理U-20S细胞48h后的凋亡细胞数分别为16.26±1.44%、34.45±2.55%、60.4±4.01%。U-20S细胞经姜黄素(20μmol/L、40μmol/L)处理12h后,Caspase-9,8,3的活性显著增加。
结论:
1.姜黄素能明显抑制人骨肉瘤U-20S细胞的体外增殖。
2.姜黄素可诱导人骨肉瘤U-20S细胞发生凋亡。
3.姜黄素诱导人骨肉瘤U-20S凋亡可能与Caspase-9,8,3的活性增加有关。
4.姜黄素为临床上骨肉瘤的治疗提供了一种新方法。
第二部分姜黄素对人骨肉瘤多药耐药的逆转作用研究
目的:观察姜黄素(Cur)对多药耐受糖蛋白(P-gp)介导的人骨肉瘤U-20S/ADM细胞多药耐药(MDR)的逆转作用。
方法:MTT法观察MDR的逆转;流式细胞仪检测细胞内罗丹明-123积聚和外排的影响;RT-PCR方法检测细胞内MDR1基因变化水平;Westen blot方法检测细胞内P-gp的变化水平。
结果:
1.MTT显示姜黄素20μmol/L能增加不同浓度阿霉素对U-20S/ADM细胞的抑制作用(P＜0.01),且姜黄素对阿霉素为2.0μg/ml时的U-20S/ADM细胞抑制作用增加幅度达49%。
2.FCM结果显示,姜黄素能增加阿霉素对U-20S/ADM的细胞毒性作用,且呈剂量依赖关系。
3.RT-PCR检测显示,耐药细胞株U-20S/ADM中MDR1的表达较U-20S增加,姜黄素对U-20S/ADM细胞MDR1的表达无明显影响。
4.Western blot结果显示,耐药细胞株U-20S/ADM中P-gp的表达较U-20S增加,姜黄素对U-20S/ADM细胞P-gp的表达无明显影响。
结论:
1.姜黄素可有效逆转U-20S/ADM细胞的多药耐药现象,其机制并非阻止MDR1基因的扩增及降低P-gp的表达,而可能是抑制耐药细胞膜上P-gp的功能。
2.MDR1基因及P-gp在骨肉瘤阿霉素耐药株中表达明显增加,可能介导了骨肉瘤的多药耐药。
Background:Osteosarcoma is the most common type of bone malignant tumor,which onset mostly between children and adolescent. Prognosis of osteosarcoma is very poor for recurrence and metastasis. Treatment of osteosarcoma includes surgery,chemotherapy,radiotherapy and Integrated treatment.The application of neoadjuvant chemotherapy has improved cure rates remarkably.Further studies have shown that multidrug resistance(MDR)to chemotherapeutic agents is a major barrier to the successful treatment of osteosarcoma.To explore new and effective chemotherapeutics will conduce to the therapy of osteosarcoma.
Curcuma is a kind of Chinese traditional medicine in common use, it has been widely used as a spice,to color cheese and butter,as a cosmetic,and in some medicinal preparations.Curcumin is a kind of hydroxybenzene pigment picked-up from curcuma,it is the major effective component,and it has various medical functions,such as antiinflammatory,antioxidant,anticoagulation,anti-artherosclerosis and anticarcinogenic effects etc,especially as a kind of well-perspectived anti-tumor drug,more and more researches have been attached on it,and it has be,come a hotspot in recent years.The mechanism of curcumin is extensive,thereinto,people pay more attention to its inducing tumor cell apoptosis.Many studies have showed that,curcumin can prevent or reduce the growth and proliferation of many kinds of tumor cells cultured in vitro,and induce apoptosis of tumor cells,such as prostatic carcinoma, nasopharyngeal carcinoma,gastric carcinoma,colon carcinoma, hepatoma,etc.Simultaneouly,curcumin can reverse MDR mediated by p-glycoprotein.But few research about curcumin's function to osteosarcoma has been done.
In the present study,the proliferation of U-2OS cells was measured by MTT assay;the morphological changes and percentage of apoptotic nuclei in U-2OS cells treated with curcumin were assayed by Hoechst 33258 staining,and apoptosis was determined by Annexin V/PI double staining technique and flow cytometry.The activities of caspase-9,8 and 3 were analyzed by Caspase Colorimetric Assay Kit.To explore the molecular mechanisms of curcumin to U-2OS or U-2OS/ADM.
PartⅠThe effects of curcumin on human osteosarcoma U-2OS cell line in vitro
Objectives:To observe the effects of curcumin on cell growth,and apoptosis in U-2OS cells,and explore the molecular mechanisms of curcumin on human osteosarcoma.
Methods:The proliferation of U-2OS cells was measured by MTT assay.The morphological changes and percentage of apoptotic nuclei in U-2OS cells treated with curcumin were assayed by Hoechst 33258 staining,and apoptosis was determined by Annexin V/PI double staining technique and flow cytometry.The activities of caspase-9,8 and 3 were analyzed by Caspase Colorimetric Assay Kit.
Results:(1)Curcumin significantly inhibited the proliferation of PC-3 cells in a concentration-and time-dependent manner.
(2)A morphological change was observed in U-2OS cells.After 24 or 48h treatment with curcumin(20μmol/L),condensation of chromatin occurred,and blebbing nuclei and granular apoptotic bodies appeared. The results of FACS showed that the percentage of apoptotic cells in the PC-3 cells treated with curcumin(20μmol/L)for 12,24,or 48 h were (13.81±1.07)%,(27.03±2.32)%or(40.76±3.96)%,respectively.After exposure to different concentrations of curcumin(10,20,or 40μmol/L) for 48 hours,the U-2OS cells occured a lot of apoptosis cells,and percentage of apoptotic nuclei respectively were(11.26±3.03)%, (32.15±2.88)%or(55.24±4.40)%.Similarly,the results of FACS also indicated a dramatic increased in apoptosis cells,and the percentage of apoptotic cells were(16.26±1.44)%,(34.45±2.55)%or(60.46±4.01)%, respectively.The activation of Caspase-9,8 or 3 were markedly increased in U-2OS cells treated with curcumin(20 or 40μmol/L)at indicated periods.
Conclusions:(1)Curcumin can significantly inhibit the proliferation of U-2OS cells.
(2)Curcumin can induce apoptosis of U-2OS cells.
(3)The effects of curcumin on induction of apoptosis may be related to the increase of Caspase-9,8 or 3' activity in U-2OS cells.
(4)Curcumin provide a novel theory and method in the therapy for human osteosarcoma clinically.
PartⅡThe reverse effects of curcumin on MDR of human osteosarcoma cell line in vitro
Objectives:To investigate the reversion of P-gp mediated multidrug resistance of U-2OS/ADM cells with curcumin in vitro.
Methods:The reversal efficacy of curcumin was measured by MTT assay.The effects of curcumin on Rh-123 uptake and efflux were analyzed by flow cytometer.RT-PCR technique was used to examine the MDR1 mRNA level,P-gp was detected by Western blot analysis.
Results:(1)MTT demonstrates curcumin(20μmol/L)can increase the cytotoxicity of Adriamycin to U-2OS/ADM cells.
(2)The result of FCM shows that Curcumin can increase the accumulation of Rh-123 and increase the cytotoxicity of Adriamycin to U-2OS/ADM cells in a dose-dependent manner.
(3)The mRNA expression of MDR1 in U-2OS/ADM cells was more higher than that in U-2OS cells,and with few influence on it when treated with curcumin(10,20 or 30μmol/L)for 48 hours by RT-PCR.
(4)The protein expression of P-gp in U-2OS/ADM cells was more higher than that in U-2OS cells,and with few influence on it when treated with curcumin(10,20 or 30μmol/L)for 48 hours by Western blot.
Conclusions:(1)The reversal mechanism of curcumin was blocked the function of P-gp in U-2OS/ADM cellular membrane and have no effect on the expression of MDR1 and P-gp.
(2)The expression of MDR1 and P-gp in U-2OS/ADM cells was increased significantly,which possibly mediated MDR in osteosarcoma.
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